Somenath Chowdhury

Mapping allostery through the covariance analysis of NMR chemical shifts

Allostery is a fundamental mechanism of regulation in biology. The residues at the end points of long-range allosteric perturbations are commonly identified by the comparative analyses of structures and dynamics in apo and effector-bound states. However, the networks of interactions mediating the propagation of allosteric signals between the end points often remain elusive. Here we show that the covariance analysis of NMR chemical shift changes caused by a set of covalently modified analogs of the allosteric effector (i.e., agonists and antagonists) reveals extended networks of coupled residues. Unexpectedly, such networks reach not only sites subject to effector-dependent structural variations, but also regions that are controlled by dynamically driven allostery. In these regions the allosteric signal is propagated mainly by dynamic rather than structural modulations, which result in subtle but highly correlated chemical shift variations. The proposed chemical shift covariance analysis (CHESCA) identifies interresidue correlations based on the combination of agglomerative clustering (AC) and singular value decomposition (SVD). AC results in dendrograms that define functional clusters of coupled residues, while SVD generates score plots that provide a residue-specific dissection of the contributions to binding and allostery. The CHESCA approach was validated by applying it to the cAMP-binding domain of the exchange protein directly activated by cAMP (EPAC) and the CHESCA results are in full agreement with independent mutational data on EPAC activation. Overall, CHESCA is a generally applicable method that utilizes a selected chemical library of effector analogs to quantitatively decode the binding and allosteric information content embedded in chemical shift changes.

Ultrasensitive determination of cysteine based on the photocurrent of nafion-functionalized CdS-MV quantum dots on an ITO electrode.

As a fundamental building block for functional and structural components of many proteins and enzymes, cysteine, a sulfur-containing nonessential amino acid, plays a critical role in many biological processes. [ 1 ] This critical residue helps to fold and maintain a stable structure of protein, contributes towards enzymatic reactions and detoxifi cation processes, and participates in numerous posttranslational modifi cations. [ 2 ]

Entropy-driven cAMP-dependent Allosteric Control of Inhibitory Interactions in Exchange Proteins Directly Activated by cAMP

Exchange proteins directly activated by cAMP (EPACs) are guanine nucleotide-exchange factors for the small GTPases Rap1 and Rap2 and represent a key receptor for the ubiquitous cAMP second messenger in eukaryotes. The cAMP-dependent activation of apoEPAC is typically rationalized in terms of a preexisting equilibrium between inactive and active states. Structural and mutagenesis analyses have shown that one of the critical determinants of the EPAC activation equilibrium is a cluster of salt bridges formed between the catalytic core and helices α1 and α2 at the N terminus of the cAMP binding domain and commonly referred to as ionic latch (IL). The IL stabilizes the inactive states in a closed topology in which access to the catalytic domain is sterically occluded by the regulatory moiety. However, it is currently not fully understood how the IL is allosterically controlled by cAMP. Chemical shift mapping studies consistently indicate that cAMP does not significantly perturb the structure of the IL spanning sites within the regulatory region, pointing to cAMP-dependent dynamic modulations as a key allosteric carrier of the cAMP-signal to the IL sites. Here, we have therefore investigated the dynamic profiles of the EPAC1 cAMP binding domain in its apo, cAMP-bound, and Rp-cAMPS phosphorothioate antagonist-bound forms using several 15N relaxation experiments. Based on the comparative analysis of dynamics in these three states, we have proposed a model of EPAC activation that incorporates the dynamic features allosterically modulated by cAMP and shows that cAMP binding weakens the IL by increasing its entropic penalty due to dynamic enhancements.

Asymmetric synthesis of amines using tert -butanesulfinamide

Chiral amines are prevalent in many bioactive molecules, including amino acids and pharmaceutical agents. tert-Butanesulfinamide (tBS) is a chiral amine reagent that has enabled the reliable asymmetric synthesis of a very broad range of different amine structures from simple, readily available starting materials. Three steps are commonly applied to the asymmetric synthesis of amines: (i) condensation of tBS with a carbonyl compound, (ii) nucleophile addition and (iii) tert-butanesulfinyl group cleavage. Here we demonstrate these steps with the preparation of a propargylic tertiary carbinamine, one of a class of amines that have been used for many different biological purposes, including click chemistry applications, diversity-oriented synthesis, the preparation of peptide isosteres and the development of protease inhibitors as drug candidates and imaging agents. The process described here can be performed in 3–4 d.

Design of a highly selective quenched activity-based probe and its application in dual color imaging studies of cathepsin S activity localization.

The cysteine cathepsins are a group of 11 proteases whose function was originally believed to be the degradation of endocytosed material with a high degree of redundancy. However, it has become clear that these enzymes are also important regulators of both health and disease. Thus, selective tools that can discriminate between members of this highly related class of enzymes will be critical to further delineate the unique biological functions of individual cathepsins. Here we present the design and synthesis of a near-infrared quenched activity-based probe (qABP) that selectively targets cathepsin S which is highly expressed in immune cells. Importantly, this high degree of selectivity is retained both in vitro and in vivo. In combination with a new green-fluorescent pan-reactive cysteine cathepsin qABP we performed dual color labeling studies in bone marrow-derived immune cells and identified vesicles containing exclusively cathepsin S activity. This observation demonstrates the value of our complementary cathepsin probes and provides evidence for the existence of specific localization of cathepsin S activity in dendritic cells.

Identification of Multiple Structurally Distinct, Nonpeptidic Small Molecule Inhibitors of Protein Arginine Deiminase 3 Using a Substrate-Based Fragment Method

The protein arginine deiminases (PADs) are a family of enzymes that catalyze the post-translational hydrolytic deimination of arginine residues. Four different enzymologically active PAD subtypes have been characterized and exhibit tissue-specific expression and association with a number of different diseases. In this Article we describe the development of an approach for the reliable discovery of low molecular weight, nonpeptidic fragment substrates of the PADs that then can be optimized and converted to mechanism-based irreversible PAD inhibitors. The approach is demonstrated by the development of potent and selective inhibitors of PAD3, a PAD subtype implicated in the neurodegenerative response to spinal cord injury. Multiple structurally distinct inhibitors were identified with the most potent inhibitors having >10,000 min(-1) M(-1) k(inact)/K(I) values and ≥10-fold selectivity for PAD3 over PADs 1, 2, and 4.

Synthesis, structure and electrochemistry of ferrocene?peptide macrocyclesElectronic supplementary information (ESI) available: Figures and tables of spectroscopic results for compounds 5?9. See http://www.rsc.org/suppdata/dt/b4/b401039f/

Redox active cyclopeptides Fc[CSA]2 (5), Fc[Gly-CSA]2 (6), Fc[Ala-CSA]2 (7), Fc[Val-CSA](2) and Fc[Leu-CSA]2 (9) (CSA = cysteamine) which are formed by the reaction of ferrocenedicarboxylic acid with peptide cystamines at high dilutions. These systems exhibit H-bonding involving the amide NH in solution as shown by their temperature dependent NMR spectra. With the exception of 5, the ferrocene macrocycles display intramolecular N...O cross-ring H-bonding in the solid state involving the amino acids proximal to the ferrocene.

Electrodeposition of ferrocenoyl peptide disulfides

Using electrodeposition of cyclic and acyclic Fc-peptide disulfides tightly-packed Fc-peptide monolayers were conveniently formed, which exhibit significant differences in their electron transfer kinetics.